Biomedical Engineering Reference
In-Depth Information
1.1 The miRNA-Protein Complex
Functional miRNA is a miRNA-ribonucleoprotein complex, also known as the
miRNA- RNA-induced silencing complex (miRNA-RISC). The miRNA
duplex, after Dicer activity, unwinds and binds to Argonaute (Ago) protein to
form the core of the functional effector complex of miRNA, the miRNP (Mour-
elatos et al., 2002). Although the events that orchestrate this loading of miRNA
onto Ago protein are still not very clear in mammals, studies from various model
systems have provided crucial insight into this process. Studies from Drosophila
melanogaster have elucidated a thermodynamic basis for the determination of
which of the two miRNA strands will actually be in complex with Ago. Accord-
ing to this model, the strand with lower thermodynamically stable base pairing
with the opposite strand on its 5
0
end is the mature miRNA (Tomari et al., 2004;
Tomari and Zamore, 2005). In flies, this thermodynamic stability is sensed by a
heterodimer of Dcr-2/R2D2, where R2D2 have selectively higher affinity toward
the more stable end of the duplex (Tomari et al., 2004).
Further processing/removal of the non-miRNA strand from the duplex and
loading of the miRNA into Ago protein is orchestrated through a bypass
mechanism, the details of which are still unclear. There are four Argonaute
families of proteins in mammals (AGO1-AGO4), and only AGO2 has been
shown to be functional inRNA interference. The RNaseH-like PIWI (P-element-
induced wimpy testis) domain of AGO2 is capable of cleaving the target RNA at
the center of the siRNA-mRNA paired region (Liu et al., 2004; Meister et al.,
2004b; Pillai et al., 2004). Apart fromArgonaute, the miRNP complex consists of
other proteins that may facilitate different aspects of miRNA function (Peters
and Meister, 2007). The interactions between miRNP complex and its target
mRNA are driven by specific base-pairing interactions between miRNA and
sequences located in the 3
0
UTR of target mRNA. The nucleotides between
Fig. 1 MicroRNA biogenesis and RISC assembly. A series of post-transcriptional processing
events are involved in biogenesis of miRNAs. The microprocessor complex (Drosha (RNase
III type of endonuclease) and DGCR8) recognizes the hairpin structures formed by pri-
miRNAs and produce an approximately 70-nucleotides hairpin known as pre-miRNA.
Notable deviation from this step of biogenesis is ''mirtrons,'' a class of miRNAs which do
not require microprocessor complex and are processed by the splicing machinery. The end
product of spliced out introns (mirtrons) have hairpin structures similar to that of pre-
miRNAs. These pre-miRNAs (
70 nucleotides in length) are then transported to cytoplasm
by exportin-5, where they are processed to
22 bp miRNA duplex by Dicer-TRBP complex.
These 22 bp duplexes have 3
0
overhangs of 2 nucleotides. One strand of these duplexes is
selectively assembled into RISC (RNA-Induced Silencing Complex), while the other strand is
degraded. Both Dicer-mediated cleavage and RISC assembly can be coupled, although this
step is not clear at the moment. This ribonucleoprotein complex (RISC) is the functional unit
and participates in miRNA-mRNA hybrid duplex formation. The miRNP-mediated regula-
tion of gene expression involves blockade of translation inhibition, blockade of translation
elongation, mRNA deadenylation and sequestration of mRNA-miRNP complex into
P-bodies or stress granules
